Hematopoietic stem cells (HSCs) are rare cells within human bone marrow that are responsible for the curative effects of hematopoietic cell transplantation (HCT). A major goal of regenerative medicine is to instruct formation of HSCs in vitro from pluripotent precursors. Despite decades of efforts, this goal has not been achieved. An improved understanding of the native mechanisms of HSC specification in the vertebrate embryo is thus required to inform these approaches. Recent studies have conclusively demonstrated that HSCs arise from hemogenic endothelium, a special population of endothelial cells within the ventral wall of the dorsal aorta that transdifferentiate into HSCs. In this application, we will utilize the unique advantages of the zebrafish embryo to provide a better understanding of how HSCs are specified genetically. Our preliminary findings demonstrate that signaling via fibroblast growth factors (FGFs) regulates HSC development at multiple stages. FGF signaling appears to be required initially to instruct HSCs from posterior lateral mesoderm (PLM), and to subsequently regulate their emergence from ventral aortic endothelium. Dissection of the mechanisms involved will lead to improved insight into the molecular cues that specify hemogenic endothelium, the birthplace of HSCs across vertebrate phyla. Our findings will ultimately provide the means to replicate human HSC development in vitro from pluripotent precursors in a precise, robust, and reproducible manner, requirements for eventual clinical utility.